Studies of glycosphingolipids in infection, immunity and differentiation

John Benktander

Department of Medical Biochemistry and Cell Biology

Institute of Biomedicine

Sahlgrenska Academy at University of Gothenburg

Gothenburg 2015

Cover illustration: MS2 and MS3 spectra of a branched Ley/H type 1 undecasaccharide (Fucα2Galβ4(Fucα3)GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glc).

Studies of glycosphingolipids in infection, immunity and differentiation © John Benktander 2015 john.benktander@gu.se

ISBN 978-91-628-9321-7 http://hdl.handle.net/2077/38348 Printed in Gothenburg, Sweden 2015 by Kompendiet, Aidla Trading AB

Abstract Cell surface glycoconjugates play a role in many biological processes such as responses to microbial infections, cell-cell interactions, differentiation, and inflammatory responses. The present work is focused on structural characterization of glycosphingolipids with potential roles in adhesion of Helicobacter pylori and Vibrio cholerae, differentiation of human pluripotent stem cells, and as blood group determinants.

In the first study, the structural binding requirements of Helicobacter pylori BabA adhesin revealed a different carbohydrate binding potential than previously defined. Adhesion of H. pylori generalist, specialist and BabA deletion mutant strains were examined using mixtures of glycosphingolipids. An unexpected binding by specialist and generalist H. pylori to the hexaosylceramide region of porcine intestinal non-acid glycosphingolipids was found. After isolation and characterization by mass spectrometry and proton NMR, the binding-active glycosphingolipid was determined as Globo H hexaosylceramide (H type 4). Further binding studies demonstrated that the generalist strain, but not the specialist strain, also recognized Globo A heptaosylceramide (A type 4). Non-secretors have an increased risk of peptic ulcer disease although they express little or no H type 1 sequences, and thus no Leb. However, these individuals have a functional FUT1 enzyme that may produce the Globo H sequence, suggesting that Globo H hexaosylceramide might have a role in H. pylori adhesion to the gastric epithelium of non-secretor individuals.

In the second study the carbohydrate binding potential of Vibrio cholerae was investigated. Binding-active glycosphingolipids, detected by the thin-layer chromatogram binding assay, were isolated and characterized by antibody binding, mass spectrometry and proton NMR. Thereby, three different binding modes were identified; the first was complex glycosphingo-lipids with GlcNAcβ3Galβ3/4GlcNAc sequence, the second glycosphingolipids with terminal Galα3Galα3Gal sequence, and the third lactosylceramide and related glycosphingolipids.

V. cholerae with non-functional chitin binding protein GbpA bound to glycosphingolipids in the same manner as the wild type bacteria, demonstrating that the GbpA is not involved in glycosphingolipid recognition.

In the third study the non-acid glycosphingolipids of human embryonic stem cells were structurally characterized. Chromatogram binding assays, mass spectrometry and proton NMR demonstrated the presence of type 2 core chain glycosphingolipids (neolactotetraosyl-ceramide, H type 2 pentaosylceramide, Lex pentaosylceramide, and Ley hexaosylceramide), and blood group A type 1 hexaosylceramide, along with the previously characterized glycosphingolipids with type 1 and type 4 core chains. Thus, the glycosphingolipid diversity of human embryonic stem cells is more complex than previously appreciated.

The PX2 antigen is assumed to belong to the GLOB blood group system and has until further notice been assigned to that blood group. However the enzymatic machinery involved in PX2 synthesis has not been determined. In the fourth study, glycosphingolipids isolated from blood group AP1

k erythrocytes, App erythrocytes and B3GALNT1 transfected MEG-01 cells were

characterized by antibody binding and mass spectrometry. The B3GALNT1 transfected MEG-01 cells had an increased expression of PX2. No P antigen or PX2 were found in the AP1

k erythrocytes, while the App erythrocytes expressed PX2, but no P1 and P antigens. The conclusion from these experiments is that the P synthase also is responsible for synthesis of the PX2 antigen.

Keywords: Glycosphingolipids, mass spectrometry, Helicobacter pylori BabA, Vibrio cholerae, human embryonic stem cells, glycosyltransferase, PX2.

Sammanfattning på svenska Glykokonjugat på ytan av celler deltar i många biologiska processer, såsom cell-cellinteraktioner, differentiering, inflammation samt infektioner. Målet med denna avhandling har varit att karaktärisera strukturen hos glykosfingolipider som har potentiell betydelse för bindning av Helicobacter pylori och Vibrio cholerae till sina målvävnader, differentiering av humana pluripotenta stamceller, samt som blodgruppsantigen.

I den första delstudien studerades bindningsspecificiteten hos BabA adhesinet från ”magsårsbakterien” Helicobacter pylori. Initialt studerades bindning av specialist, generalist, och BabA knock-out H. pylori stammar till glykosfingolipidblandningar. Därvid upptäcktes en bindning av specialist och generalist H. pylori till en neutral hexaosylceramid från gristarm. Den bindningsaktiva glykosfingolipiden isolerades och karaktäriserades med masspektrometri och proton NMR som Globo H hexaosylceramid (H typ 4). Ytterligare bindningsstudier visade att generalist H. pylori, men inte specialist H. pylori, också binder till Globo A heptaosylceramide (A type 4). Förmågan att binda till blodgruppsantigen på typ 4-kedjor kan ha betydelse för adhesion till magsäckspeitelet hos ”non-secretor” individer, vilka har en förhöjd risk att utveckla magsårssjukdom.

I nästa studie gjordes en kartläggning av Vibrio cholerae-bindande glykosfingolipider. Flera interaktioner med komplexa neutrala glykosfingolipider påvisades vid screening av glykosfingolipider från olika källor. Efter isolering och karaktärisering av bindningsaktiva glykosfingolipider med masspektrometri och proton NMR fastställdes tre olika bindnings-mönster. Den första var bindning till komplexa lakto/neolakto strukturer, det andra glykosfingolipider med en terminal Galα3Galα3Gal sekvens, och den tredje laktosylceramid och relaterade glykosfingolipider. Det kitin-bindande proteinet GbpA från V. cholerae är ett potentiellt adhesin. Dock visade bindningsstudier med mutant V.cholerae med inaktiverat GbpA att detta protein inte är involverat i bindning till glykosfingolipider.

I den tredje studien karaktäriserades de neutrala glykosfingolipiderna från humana embryonala stamceller. Flera glykosfingolipider med typ 2 kedja, såsom neolaktotetraosyl-ceramid, H typ 2 pentaosylceramid, Lex pentaosylceramid och Ley hexaosylceramid, identifierades. En A typ 1 hexaosylceramid påvisades också, liksom tidigare beskrivna glyko-sfingolipider med typ 1 och typ 4 kedjor. Vidare karaktäriserades glykosfingolipider med kort kolhydratkedja som galaktosylceramid, glukosylceramid, laktosylceramid, galabiosylceramid, globotriasylceramid och laktotriasylceramid. Detta visade att uttrycket av glykosfingolipider i humana embryonala stamceller är mer komplext än man tidigare ansett.

I den fjärde undersöktes den genetiska bakgrunden till blodgruppsantigenet PX2. För att testa detta studerades uttrycket av glykosfingolipider i humana erytrocyter med blodgrupperna App och AP1

k, samt i celler transfekterade med genen för glykosyltransferaset P synthase (B3GALNT1). Glykosfingolipiderna karaktäriserades med antikroppsbindning och masspektrometri. Transfektion med B3GALNT1 gav ett förhöjt uttryck av PX2. I AP1

k erytrocyterna saknades P och PX2 antigen, medan App erytrocyterna uttryckte PX2, men inte

P1 and P antigen. Sammanlagt visar detta att det B3GALNT1-kodade P-syntaset även producerar PX2 antigenet.

List of papers

I. Benktander J, Ångström J, Breimer ME, Teneberg S. Re-definition of the

carbohydrate binding specificity of Helicobacter pylori BabA adhesin. 2012. J Biol Chem 287, 31712-24.

II. Benktander J, Ångström J, Karlsson H, Teymournejad O, Lindén S, Lebens M, Teneberg S. The repertoire of glycosphingolipids recognized by Vibrio cholerae. 2013. PLoS One, 8, e53999.

III. Barone A, Benktander J, Ångström J, Aspegren A, Björquist P, Teneberg S,

Breimer ME. Structural complexity of non-acid glycosphingolipids in human embryonic stem cells grown under feeder-free conditions. 2013. J Biol Chem 288, 10035-50.

IV. Westman JS*, Benktander J*, Storry JR*, Peyrard T, Hult AK, Hellberg A,

Teneberg S, Olsson ML. Genetic basis of PX2, a recently acknowledged glycolipid blood group antigen. Submitted.

* Contributed equally to this study.

Contents 1. Abbreviations ...................................................................................................................................... 1

2. Background ......................................................................................................................................... 2

2.1. Glycobiology ................................................................................................................................ 2

2.1.1. Glycosphingolipids ................................................................................................................ 3

2.1.2. Glycosphingolipid biosynthesis ............................................................................................. 4

2.1.3. Functions of glycosphingolipids ............................................................................................ 6

2.2. Microbial glycan adhesion ........................................................................................................... 6

2.2.1. Helicobacter pylori ................................................................................................................ 6

2.2.2. Vibrio cholerae ...................................................................................................................... 7

2.3. Human embryonic stem cells ....................................................................................................... 8

2.4. The P1PK and GLOB blood group systems ................................................................................. 8

3. Aims .................................................................................................................................................. 10

4. Methods ............................................................................................................................................. 11

4.1. Extraction and purification of glycosphingolipids ..................................................................... 11

4.2. Structural characterization of glycosphingolipids ...................................................................... 13

4.2.1. Thin-layer chromatography, detection and binding assays ................................................. 13

4.2.2. Mass spectrometry ............................................................................................................... 13

4.2.3. Mass spectrometry of glycosphingolipids ........................................................................... 15

4.2.4. Nuclear magnetic resonance spectroscopy .......................................................................... 16

4.3. Flow cytometry........................................................................................................................... 17

5. Results and discussion ....................................................................................................................... 18

5.1. Glycosphingolipids in infection ................................................................................................. 18

5.1.1. Paper I: Re-definition of the carbohydrate binding specificity of Helicobacter pylori BabA adhesin. .......................................................................................................................................... 18

5.1.2. Paper II: The repertoire of glycosphingolipids recognized by Vibrio cholerae. ................. 21

5.2. Glycosphingolipids in differentiation ......................................................................................... 23

5.2.1. Paper III: Structural complexity of non-acid glycosphingolipids in human embryonic stem cells grown under feeder-free conditions. ..................................................................................... 23

5.3. Glycosphingolipids in immune recognition ............................................................................... 26

5.3.1. Paper IV: Genetic basis of PX2, a recently acknowledged glycolipid blood group antigen. ....................................................................................................................................................... 26

6. Future perspectives ............................................................................................................................ 31

7. Acknowledgements ........................................................................................................................... 32

8. References ......................................................................................................................................... 34

1. Abbreviations BabA Blood group antigen-binding adhesion Cer Ceramide ESI Electrospray ionization Fuc Fucose Gal Galactose GalNAc N-acetylgalactosamine Glc Glucose GlcNAc N-acetylglucosamine hESC Human embryonic stem cells Hex Hexose HexNAc N-acetylhexosamine LacCer Lactosylceramide LC/MS Liquid chromatography mass spectrometry MS Mass spectrometry MS/MS and MS2 Tandem mass spectrometry m/z Mass-to-charge NeuAc N-acetylneuraminic acid/Sialic acid NMR Nuclear magnetic resonance RT Retention time

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2. Background 2.1. Glycobiology

Glycobiology encompasses the studies of glycans in all its forms. A glycan, or the more common name carbohydrate, is a molecule composed of carbon, hydrogen and oxygen. The simplest glycan unit is a monosaccharide. When monosaccharides are joined together to form saccharide chains they are referred to as oligosaccharides, while those having >10 saccharide units are called polysaccharides.

Figure 1: Mammalian cell membrane glycoconjugates (Modified from Fuster and Esko, 2005, [1]) with symbols and abbreviations of common monosaccharides [2].

Glycans can be found as free saccharides, as in mammalian milk, but often the glycan is linked to a non-carbohydrate moiety. Those structures are generally named glycoconjugates. There are many types of glycoconjugates, such as glycoproteins, glycolipids and proteoglycans (Figure 1).

The major types of glycoproteins comprise N- and O-glycans. The glycans are directly attached to the amino acids of the proteins. N-glycans are attached to asparagine side-chains. The N-glycans are derived from a dolichol-oligosaccharide precursor and are generally classified into three types; oligomannose, complex and hybrid type.

O-glycans are linked to the side-chains of serine or threonine. The majority of O-glycans consists of one of four core structures, all starting with a GalNAc attached to the protein. Mucins are heavily glycosylated O-glycan proteins.

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Finally, the proteoglycans are heavily glycosylated proteins. Large glycosaminoglycan (GAG) chains are linked to serine via a tetrasaccharide linker. The proteoglycan proteins have a wide variety of numbers of glycosaminoglycans attached, some only have one while others have more than 100 GAGs. The glycosaminoglycans structure is composed of repeating disaccharide units of acetylated or sulfated hexosamines, linked to either a galactose or an uronic acid. [3].

There are also different glycolipid classes, though the focus in this thesis will be on glycosphingolipids.

2.1.1. Glycosphingolipids

Glycosphingolipids are amphipathic molecules consisting of a polar carbohydrate linked to a hydrophobic ceramide moiety (Figure 2). The ceramide part anchors the glycosphingolipid into the cell membrane with the carbohydrate chain sticking out from the membrane. The ceramide consists of a long-chain base and a fatty acid. The most common long-chain bases are sphingosine, sphinganine and phytosphingosine, all with 18 carbon chains [4, 5]. In some tissues long-chain bases with 20 carbon chains are present [6]. The fatty acid chains are more variable and consist of 14-30 carbons atoms, or even longer, although fatty acids with 16-24 carbons are most commonly found. The fatty acids can also be unsaturated and have α-hydroxy groups [3].

Figure 2: Structure of a lactosylceramide d18:1-16:0 glycosphingolipid. The carbon number assignment is shown on the monosaccharide to the left. Shorthand nomenclature for fatty acids and bases: d18:1 represents a sphingosine and t18:0 represents a phytosphingosine. The number before the colon refers to the carbon chain length and the number after gives the total number of double bonds. Fatty acids with a 2-hydroxy group are denoted by the prefix h.

Glycosphingolipids are heterogeneously distributed in the outer leaflet of the cell membrane due to stabilizing hydrogen bounds. They are therefore assembled into a formation called “lipid rafts” containing sphingolipids, cholesterol and specific membrane proteins [7]. There are also glycosphingolipids assembling without cholesterol in microdomains called “glycosynapses”, that are involved in carbohydrate dependent interactions [8].

Glycosphingolipids is generally characterized as either acid or non-acid. The main acid glycosphingolipids are of two types; the gangliosides, which have sialic acids and the sulfated glycosphingolipids, where the saccharide is modified with sulfate.

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The non-acid glycosphingolipids includes all glycosphingolipids that have no charged moieties and the majority of the oligosaccharides are constructed from one of the major core-structures. Some of the vertebrate major glycosphingolipid core structures are shown in Table 1. Table 1: Common core structures of vertebrate glycosphingolipids.

Core structure series Abbreviation Structure Lacto Lc Galβ3GlcNAcβ3*Galβ4GlcβCer Neolacto nLc Galβ4GlcNAcβ3Galβ4GlcβCer Globo Gb GalNAcβ3Galα4Galβ4GlcβCer Isoglobo iGb GalNAcβ3Galα3Galβ4GlcβCer Ganglio Gg Galβ4GalNAcβ4Galβ4GlcβCer

* The carbohydrate sequence defining each core is underlined.

Another glycosphingolipid core is the athro sequence (GalNAcβ4GlcNAcβ3Manβ4GlcβCer), found in several classes of invertebrates [3]. Different types of glycosphingolipids are expressed differently depending on species [9], individual [10], tissue and cell type [11].

2.1.2. Glycosphingolipid biosynthesis

The synthesis of the glycosphingolipids starts at the cytoplasmic side of the Golgi apparatus [12, 13] where the GlcCer synthase adds a glucose in β-linkage to the hydroxyl group at position 1 of the ceramide. The ceramide is synthesized earlier in the endoplasmic reticulum and is then transported to the Golgi [14]. Further additions of glycans to the glucosylceramide occurs after switching to the luminal side of the Golgi [15] with the help of an uncharacterized flippase [16]. In some cases a galactose is added to the ceramides. This happens at the luminal side in the endoplasmic reticulum [17], though the downstream synthesis pathway is very limited for galactosylceramide, therefore this pathway will not be further described.

After synthesis of glucosylceramide, a galactose is added in the Golgi lumen by the LacCer synthase. The generated lactosylceramide has multiple possible synthesis pathways, one is where the Gb3 synthase adds a galactose in α4-linkage to lactosylceramide creating the globotriaosylceramide, which is the precursor of the globo-series glycosphingolipids. The iGb3 synthase is also a possible synthesis pathway, where the iGb3 synthase transfers a galactose in a α3-linkage, leading to the isoglobo-series. This is however not normally found in humans since the gene of the iGb3 synthase is inactivated [18]. The precursor of the ganglio-series, gangliotriasylceramide, is another possible synthesis, where GM2/GM3 synthase add a β4-linked N-acetylgalactosamine to lactosylceramide. The lactosylceramide can also be substituted with N-acetylglucosamine in a β3-linkage by the Lc3 synthase, generating the lactotriaosylceramide glycosphingolipid [19]. From the lactotriaosylceramide precursor the lacto-series and the neolacto-series is generated either by an addition of a Galβ3 (lacto) or a Galβ4 (neolacto) (Figure 3A).

Aside from the synthases that divides the glycosphingolipids into the different core structures, a lot of glycosyltransferases modify the terminal ends of glycosphingolipid carbohydrate chains. A common addition is fucosylation by the blood group O and Lewis enzymes (Figure

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3B). Addition of one or more sialic acids by the different sialyltransferases creates a ganglioside, while addition of a sulfate group creates a sulfated glycosphingolipid. The regulation of glycan structures expressed by a cell is not entirely understood, but is known to be influenced by kinetic parameters of the glycosyltransferases and organization in the Golgi. It is also possible that glycans are regulated by transcriptional regulation of the transferases. However, to express a specific glycan the right combination of glycosyltransferases must be expressed [20].

Figure 3: A) Simplified overview of synthesis pathways for the basic core structures of the glycosphingolipids. The glycosphingolipid synthesis is initiated by the addition of either a Gal or a Glc to the ceramide. The enzymes responsible for this are the GalCer synthase (UDP-galactose:ceramide galacosyltransferase) and GlcCer synthase (UDP-glucose:ceramide glycosyltransferase). The LacCer synthase (β-1,4-galactosyltransferase 5) continues by transferring a Gal to the glucosylceramide. From lactosylceramide, several synthesis pathways are possible which generates the precursors of the different core-structure series. The Lc3 synthase (β-1,3-N-acetylglucosaminyltransferases 5) transfers a GlcNac to the lactosylceramide, thus creating the precursor of the lacto/neolacto series. Another possibility is that the GM2/GM3 synthase (β-1,4-N-acetyl-galactosaminyltransferase) adds a GalNAc creating the gangliotriaosylceramide. Aside from these the Gb3 synthase (α-1,4-galactosyltransferase) and iGb3 synthase (α-1,3-galactosyltransferase) can transfer a Gal in a α4- or α3-linkage, respectively, initiating the globo-series and the isoglobo-series [reviewed in [19]. B) Basic overview of the synthesis of the ABO and Lewis blood group systems. The FUT3 gene encodes the Lewis enzyme (fucosyltransferase 3) that is an α-1,3/4-fucosyltransferase that adds a fucose to a type 1 or type 2 chains. However, several fucosyltransferases, such as FUT4, FUT5, FUT6 and FUT7, can also act on the type 2 chain and produce the Lewis antigens [21]. There are two genes responsible for the creation of the H antigen, the secretor gene and the H gene. The secretor gene encodes the FUT2 enzyme that adds Fucα2 to both type 1 and type 2 chains, while the H gene that encodes the FUT1 enzyme, which preferably adds Fucα2 to type 2 chains. The A transferase (α-1,3-N-acetyl-galactosaminyltransferase) and B transferase (α-1,3-galactosyltransferase) adds GalNAcα3 and Galα3, respectively, to the galactose in the H antigen (both type 1 and 2) [22].

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2.1.3. Functions of glycosphingolipids

The specific function of each glycosphingolipid is generally hard to define since they often have widespread functions, which overlap with other glycosphingolipids and components. One hypothesis is that the basic function of the glycosphingolipids is as a low-cost method of protecting the cell membrane with a carbohydrate layer (a.k.a. glycocalyx) [23]. According to this hypothesis the glycosphingolipids can be divided into two groups according to function; those who give the cell a protective carbohydrate coat with practically no other function, and those who also have a specified function.

Another hypothesis [24] attempts to explain the variety of glycosphingolipids as the result of an evolution in response to host-pathogen or host-symbiont interactions that drives the organisms to express new glycotransferases, capping the glycans with new saccharides. Other secondary functions are believed to have appeared later in evolution.

The gangliosides have more defined functions, mainly connected to the nervous system, cell-cell adhesion and recognition [25]. Several phenotypical defects are found in ganglioside-synthase knock-out mice [reviewed in [26], which generally affects brain development. Less defined functions has been found in the non-acid glycosphingolipids, but some are still hinted, such as involvement in cell-stage specific signaling and cell proliferation [23].

2.2. Microbial glycan adhesion

To attach to cellular surfaces or extracellular matrices bacteria often express adhesion proteins called adhesins. In many cases the adhesins bind the glycan part of glycoconjugates such as glycoproteins (e.g. mucins) or glycolipids, which enables infection and invasion by the bacteria. Binding to carbohydrates also has the advantage of mediating infection to specific hosts and tissue types [27, 28].

Adhesins can be of different types, like fimbriae, which are organelles extending from the bacterial surface. The adhesins of bacterial fimbriae are with either the tip protein or the major structural subunit [29]. There are also adhesins which are integral cell wall proteins of the bacteria [30]. Similar to adhesins, bacterial toxins also take advantage of carbohydrate recognition. Toxins such as the Vibrio cholerae cholera toxin (CT) [31], Bacillus thuringiensis crystal toxin (Bt toxin) [32], Clostridium tetani tetanus toxin [33], Escherichia coli heat-labile enterotoxin (LT) [34], Clostridium difficile toxin A [35] and Shigella dysenteria Shiga toxin [36] bind to carbohydrate structures.

2.2.1. Helicobacter pylori

In the 1980ies Warren and Marshall discovered that a previously unknown bacterium was connected with acute and chronic gastritis [37]. The bacterium was first given the name Campylobacter pylori, which later was changed to the present name Helicobacter pylori. H. pylori infection is nowadays also associated with peptic ulcers and gastric carcinogenesis [38]. Overall 47 % of all humans are positive for H. pylori infection. An incidence between 11% and 69% is found throughout different parts of the world, with low prevalence in developed countries and high in developing countries [39] .

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H. pylori normally resides in the gastric mucus layer, though some bacteria adhere directly to the gastric epithelial cells [40]. Two H. pylori adhesins, the blood group antigen-binding adhesin BabA, and the sialic acid-binding adhesin SabA, were previously characterized [38], and recently a GalNAcβ1-4GlcNAc binding adhesin termed LabA (lacdiNAc-binding adhesin) has been found [41]. The BabA adhesin bind primarily to the Lewis b determinant (Fucα2Galβ3(Fucα4)GlcNAc-R), and also to the H type 1 determinant (Fucα2Galβ3GlcNAc-R) [42, 43]. Strains expressing not only BabA but also vacuolating cytotoxin VacA and cytotoxin-associated antigen CagA are generally associated with a more severe disease [44]. SabA binds sialylated glycan structures such as sialyl-Lex (NeuAcα3Galβ4(Fucα3)GlcNAc-R). SabA mediated adhesion is suggested to be most prevalent during chronic inflammation [45].

The BabA adhesin of H. pylori adapts to the expression of fucosylated glycans among the local population. In populations from Europe and USA where blood group A, B and O are all widespread, strains called “generalists” express a BabA that is able to bind to determinants of these blood groups. In South American native population where only blood group O is prevalent, local H. pylori BabA does not have the ability to bind A and B type epitopes. The strains with this BabA with more narrow specificity are called “specialists” [46].

2.2.2. Vibrio cholerae

Vibrio cholerae was first described in 1854, and since then the strains and their characteristics have changed considerably. There have been seven or eight cholera pandemics since 1817, before that cholera was only found in the eastern parts of the world. It is believed that the classical biotype was the cause of the six first pandemics, whereas after 1961, when the seventh pandemic began, the El Tor strain took over [47]. In 1992 in Chennai in India, a new type of strain named V. cholerae O139 Bengal was isolated from a cholera epidemic [48]. That strain was first believed to be the start of an up-coming eight cholera pandemic, but since no further outbreaks have been found, many find it unlikely. More than 200 serogroups of V. cholerae have so far been discovered, with only two of them (O1 and O139) causing contagious cholera [49]. The O1 strains are further divided into two biotypes (classical and El Tor) and three serotypes (Ogawa, Inaba and Hikojima) [50].

V. cholerae infects the small intestine after ingestion of the bacteria. A cholera infection is characterized by severe watery diarrhea which can be potentially fatal, because of dehydration and loss of electrolytes. An estimate of 3 to 5 million people is affected yearly by cholera, leading to approximately 100.000-120.000 deaths (WHO, 2012). The diarrhea is mainly caused by the cholera toxin, an AB5-toxin with one A-subunit and five B-subunits [51]. The B-subunits mediates attachment to the small intestine epithelium by the ligand ganglioside GM1 (Galβ3GalNAcβ4(NeuAcα3)Galβ4Glcβ1Cer)[52]. Cholera toxin binding to GM1 is a well-known fact, but the requirements for adhesion of V. cholerae bacterial cells to the human intestinal epithelium have until now not been investigated. A potential candidate adhesin is the chitin binding protein GbpA that has been found to bind chitin surfaces, human intestinal cells and mouse intestine [53, 54]. However, no carbohydrate structure required for GbpA binding to the human small intestine has been identified.

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2.3. Human embryonic stem cells

Embryonic stem cells have the distinguishing ability of pluripotency, meaning that the cell has the ability to transform into all cell types in the adult body. Pluripotent stem cells are also able to proliferate indefinitely while retaining their diverse transformation ability. Human embryonic stem cells (hESC) are obtained by taking cells from the inner cell mass, called epiblast or embryoblast, from the blastocyst, that is formed five days after fertilization [55, 56].

A number of carbohydrate markers are used for the identification of undifferentiated stem cells, as the SSEA-3 (stage-specific embryonic antigen 3), SSEA-4 [57], TRA-1-60 [58] and TRA-1-81 [44]. The SSEA-3 and -4 markers are glycosphingolipids (globopentaosylceramide and sialyl-globopentaosylceramide, respectively). Both of these have the globo core with the Galα4Gal structure, which is basically only found in glycosphingolipids [59].

The glycosphingolipids of stem cells has mainly been studied using mouse embryonic cells, and there were only two previous studies of the glycosphingolipids in hESC [60, 61]. These studies identified the non-acid glycosphingolipids globotetraosylceramide, globopentaosyl-ceramide (SSEA-3), globo H hexaosylceramide and H type 1 pentaosylceramide, as well as the acid glycosphingolipids GM3, GM1, GD1a or GD1b, sialyl-globopentaosylceramide (SSEA-4) and disialyl-globopentaosylceramide.

2.4. The P1PK and GLOB blood group systems

Aside from the common blood groups ABO and Rh, there are a number of minor blood group systems such as the GLOB and P1PK blood group systems. In total, 35 blood group systems have been defined (2015) [62].

The P1PK blood group system, previously called the P system, includes the P1 (Galα4Galβ4GlcNAcβ3Galβ4Glcβ1Cer) and Pk antigen (Gb3; Galα4Galβ4Glcβ1Cer). Both the Pk and P1 antigen is synthesized by the A4GALT-encoded enzyme. However, due to single-nucleotide polymorphisms in the gene, P1 is not always expressed [63]. A later addition to the P1PK blood group is the NOR antigen (Galα4GalNAcβ3Galα4Galβ4Glc-β1Cer). It was added after the discovery of a mutation were glutamic acid is replacing glutamine at amino acid position 211 in the A4GALT-encoded enzyme, inducing the expression of NOR [64, 65]. The P antigen (Gb4; GalNAcβ3Galα4Galβ4Glcβ1Cer), the predominant non-acid glycosphingolipid in human erythrocytes [66], belongs to the GLOB blood group system. This is associated with the P1K1 blood group, since its precursor is the Pk antigen, but is synthesized by the B3GALNT1-encoded P synthase [67] (Figure 4).

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Figure 4: Basic overview of P1PK and GLOB synthesis pathway. The α4Gal transferase, encoded by A4GALT, transfers a Gal to the terminal Gal of lactosylceramide and paragloboside in a α4-linkage. B3GALNT1 encodes a β3GalNAc transferase that adds a GalNAc to the terminal Gal of Pk in a β3-linkage.

Besides the P antigen, the x2 pentaosylceramide (GalNAcβ3Galβ4GlcNAcβ3Galβ4Glcβ1Cer) has been included in the GLOB blood group system, as of 2011, under the name PX2 [68]. The genetic background of PX2 is not yet known, but it was still included in the GLOB blood group until further notice.

Table 2: General outline of P phenotypes, their expressed antigens and functional genes. Phenotype Expressed antigens Functional genes P1 P, Pk, P1 A4GALT, B3GALNT1 P2 P, Pk A4GALT*, B3GALNT1 P1

k Pk, P1 A4GALT P2

k Pk A4GALT* p None B3GALNT1

* Indicates an A4GALT gene with single-nucleotide polymorphism resulting in inability to synthesize P1.

There are five different phenotypes of P1PK and GLOB blood groups; the common P1 and P2, the rare P1

k and P2k phenotypes, and the p phenotype [69]. The antigens expressed by

respective phenotype are shown in Table 2. The null phenotypes of P1PK or GLOB blood group systems have a slightly increased risk of intravascular hemolytic transfusion reactions and spontaneous abortions, caused by natural antibodies against the missing P antigens [70].

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3. Aims The overall aim of this thesis was to characterize glycosphingolipids with potential roles in microbial adhesion, differentiation of human pluripotent stem cells, and as blood group determinants. Specific aims: 1. To characterize the glycosphingolipids involved in adhesion of Vibrio cholera and

Helicobacter pylori. 2. To characterize the glycosphingolipids of human embryonic stem cells. 3. To characterize the glycosphingolipids of human blood group AP1

k and App erythrocytes, in the course of an investigation of the role of P synthase in synthesis of the x2 glycosphingolipid.

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4. Methods 4.1. Extraction and purification of glycosphingolipids

The method used for extraction and isolation of glycosphingolipids was developed by K-A. Karlsson, and has been used for over 30 years [71]. It mainly uses polarity to separate the glycosphingolipids on silic acid columns, but also applies ion-exchange chromatography. With this method it is possible to purify glycosphingolipids with glycans ranging from 1 to 18 saccharide units.

The tissues or cells are prepared for extraction by lyophilization. After this the sample is extracted with mixtures of chloroform and methanol in a Soxhlet apparatus. The extracts are then pooled and dried. Thereafter, the lipid extract is subjected to a mild alkaline methanolysis, which will break the ester bonds of phospholipids and as a result release the glycerol linked fatty acids. This is followed by dialysis for 3-5 days to remove the salt. Fatty acids, ceramide and other non-polar lipids are separated from glycosphingolipids by silicic acid column chromatography, utilizing increasing methanol to chloroform ratios. Next DEAE (diethylaminoethyl) ion exchange chromatography is used to separate acid and non-acid glycosphingolipids. Non-acid glycosphingolipid are eluted with chloroform and methanol, while the acid glycosphingolipids are eluted with an excess of salt (LiCl). Then the non-acid fraction is acetylated, giving a decreased polarity since the hydroxyl groups on the saccharides are caped with non-polar acetate groups. The acetylated glycosphingolipids are separated on a silicic acid column to remove sphingomyelin, which only gets a small change in polarity compared to the glycosphingolipids. After this the acetylated glycosphingolipid fraction is deacetylated with a mild alkaline treatment, which restores them to the original state. After dialysis, the glycosphingolipids are subjected to another DEAE were alkali-stable phospholipids with amine groups (such as plasmalogens), which have turned acidic by acetylation, are separated along with remaining acid glycosphingolipids. Finally a silicic acid column is used to remove remaining fatty acids and other non-polar molecules (Figure 5).

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Figure 5: General scheme for glycosphingolipid (GSL) purification and characterization.

Iatrobeads (Iatrobeads 6RS-8060; Iatron Laboratories, Tokyo) column chromatography is used for further separation of the glycosphingolipid fractions, since Iatrobeads have a smaller particle size which allows a more fine-tuned separation. The fractions are eluted by increasing levels of methanol to chloroform or by mobility in chloroform/methanol/water (60:35:8, by volume). The fractions collected are pooled according to their mobility on high performance thin-layer chromatography (HPTLC) plates after visualization by anisaldehyde staining. The

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fractions are also pooled according to the results from antibody, lectin and bacterial binding assays.

4.2. Structural characterization of glycosphingolipids

Structural characterization of glycosphingolipids involves definition of the types of carbohydrates, the carbohydrate sequence, the linkage positions, linkage anomerity and the ceramide composition. These data are mainly obtained by mass spectrometry, proton NMR spectroscopy, degradation studies and chromatogram binding assays. The data obtained is best interpreted in combination, due to the different information obtained from each method. Mass spectrometry is for example unable to predict the types of saccharides and anomerity of linkages, but is very good at determining carbohydrate sequences and some linkage positions. Chromatogram binding assays with specific antibodies and lectins may confirm the presence of certain carbohydrate epitopes in the samples, thus filling in the blind spots of mass spectrometry. NMR is a good method for determining the types of monosaccharides and linkage anomerity, but has more difficulty to find the correct carbohydrate sequence, therefore it works very well in combination with mass spectrometry. However, NMR is not always possible since it requires a larger quantity of pure sample.

4.2.1. Thin-layer chromatography, detection and binding assays

Thin-layer chromatography is a method to separate the glycosphingolipids depending on their polarity, and at the same time visualizing them. It can either be done on aluminium, plastic or glass-backed plates covered by silica gel [72]. Samples are eluted with mixtures of chloroform, methanol and water as mobile phase. Chemical detection of glycosphingolipids may be done with different reagents as orcinol [73], anisaldehyde [74] or resorcinol [75].

Chromatogram binding assay (CBA) is used for detecting specific epitopes/antigens of samples on thin-layer chromatograms and can therefore be used for characterization. This method may also be used for establishing the binding specificity of binding agents. In short the procedure starts by coating the dried thin-layer chromatograms in plastic. After that, it is possible to bind radiolabeled bacteria, viruses, lectins, antibodies and various proteins to the glycosphingolipids on the chromatogram [76-78]. The binding is usually visualized by a x-ray film reacting to radioactivity [79, 80], or enzyme-conjugated antibodies with visualizing substrate [81].

4.2.2. Mass spectrometry

In the late 1880ies physicists described mass spectrometry, but it was not until 1913 that Sir Joseph John Thomson, who invented the first mass spectrometry instrument, could demonstrate the use in analytical chemistry. Organic substances could be detected in the late 1940ies, and in 1952 a method for combining gas chromatography (GC) and MS was invented, which made MS useful in biochemistry. Although GC-MS is excellent when analyzing many compounds, the downside is that the molecules analyzed are required to be thermally stable, which limits the number of compounds that can be analyzed.

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The soft ionization methods, electrospray/ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) appeared in the yearly 1990ies and enabled studies on larger biomolecules such as proteins [82, 83]. These methods also supported coupling to liquid chromatography (LC) and capillary electrophoresis (CE) [84].

Mass spectrometry is basically an analytical technique creating positively or negatively charged gas-phase ions which are separated according to their mass-to-charge ratio (m/z). Electrospray ionization (ESI) is the ionization method focused on in this thesis. Electrospray is enabled by adding a voltage to the needle that holds the sample liquid. This will cause the liquid to adopt an elliptical shape. At a certain voltage the elliptical shape changes to a pointed cone, called Taylor cone [85]. This cone will generate a spray of small charged droplets. The small droplets will shrink in size due to evaporation of neutrally charged solvent molecules and charged ions will be formed when the number of charges is accumulating in the droplets. There are currently two different theories explaining the ESI ionization mechanism more in detail: the ion evaporation model and the charge residue model [86].

A linear ion trap quadrupole (LTQ) mass analyzer was used in this thesis. LTQ utilizes an electric potential to guide the ions from the electrospray into an ion transfer capillary. The ion transfer capillary leads the ions into a series of two quadrupoles and one octapole before reaching the linear ion trap. During this time, ions are transferred down a pressure gradient while increasing their kinetic energy. The ions are then trapped inside the ion trap with an electric potential which is able to eject ions of specific m/z by voltages of matching radiofrequencies. The ions ejected are detected by a detector defining the intensity of each m/z. Inside the ion trap, collision induced dissociation (CID) is managed by increasing the ion motion leading to dissociation into fragments from the parent ions, after gaining enough energy from colliding with helium gas. The fragment ions are ejected in the same manner as described before [87].

The precursor ions collided in the collision cell of the mass analyzer gives, after detection, a spectrum of the fragments called MS2 or MS/MS spectrum. A MS2 fragment can be further investigated by repeated collisions in the collision cell, creating new fragments shown in a MS3 spectrum. For glycans, MS2 mainly yields the fragment ions C, B, Z, Y and the ring-cleavage fragments A (Figure 6). For underivatized oligosaccharides, the A fragments are found in many types and indicate different linkages, as e.g. 0,2A-fragments which indicate 1-4 linkages and 0,3A-fragments which indicate 1-6 linkages [88].

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Figure 6: MS/MS fragments created in glycan analysis. R denotes the reducing end.

The ions obtained from ESI/MS in negative mode, yields the deprotonated ions [M-H+]-, were M is molecular mass. For positive mode, protonated ions [M+H+]+, lithium adducts [M+Li+]+ and sodium adducts [M+Na+]+ can be generated. Ions of high molecular weight often have more than one charge which decreases the m/z as a result, e.g. an ion which is doubly charged has half of the m/z value of a singly charged ion of the same type [89]. 4.2.3. Mass spectrometry of glycosphingolipids

The first mass spectra of a glycosphingolipid were obtained in 1970 by using electron ionization (EI) on a trimethylsilylated glucosylceramide [5]. Permethylation, i.e. chemical permethylation of the hydroxyl groups, enabled glycosphingolipids to be analysed on MS by increasing the stability and volatility of the molecules [90]. Also combined permethylation and reduction of glycosphingolipids helped in improving MS analyses [91]. In 1983 underivatized glycosphingolipids could be analyzed with the relatively soft ionization method fast atom bombardment (FAB) in negative ion mode [92]. This was further improved in the early 1990ies when ESI-MS started to be applied to glycosphingolipid MS analysis [93]. Derivatized glycosphingolipids e.g. permethylated intact glycosphingolipids are still used, but not to the same degree since many of the instruments previously used are not manufactured today. A promising technique is thin-layer chromatogram coupled mass spectrometry (TLC-MS), that uses thin-layer chromatograms to separate the glycosphingolipids, with a possibility to detect with both chemical staining and overlay assay, in combination with mass spectrometry to structurally characterize the glycosphingolipids. It can be applied as an indirect method were the thin-layer plate is scraped combined with preparation, or as a direct method were the plate is directly scanned after addition of a liquid matrix, as TLC-IR-MALDI-MS [94, 95].

An option when analyzing glycosphingolipids is to remove the ceramide part. Enzymes such as ceramide glycanases and endoglycoceramidases enable cleavage of the glycosphingolipids into ceramides and glycans. By analyzing the oligosaccharides thereby obtained, liquid chromatography separation of the different saccharides is more efficient, and more information, such as linkage positions, can be obtained [88, 96]. However, the obvious

15

disadvantage of removing the ceramide is that no information about the ceramide profile is obtained. Nevertheless, depending on the experiment, determination of the ceramide type is not always necessary [97]. Endoglycoceramidase II from Rhodococcus spp. was used for hydrolyzing glycosphingolipids in this thesis. A weakness with this enzyme that must be noted is that endoglycoceramidase II has restricted hydrolytic capacity on glycosphingolipids in the globoseries and some specific gangliosides [98]. Ideally the ceramide glycanase from Macrobdella decora would be used, which has a broader activity [99]. However this enzyme is no longer commercially available.

The glycosphingolipid-derived oligosaccharides are analyzed by capillary-LC/MS, with separation on a column packed in-house with porous graphite particles [96]. The oligosaccharides are eluted with a gradient of water to acetonitrile. The eluting saccharides are then analyzed in negative ion mode on a LTQ linear quadrupole ion trap mass spectrometer.

For analysis of native glycosphingolipids, they are first dissolved in 3:1 methanol:acetonitrile (by volume) and then separated on a column packed in-house with polyamine II particles by elution with an acetonitrile to water gradient. The eluting saccharides were analyzed in negative ion mode on a LTQ linear quadrupole ion trap mass spectrometer.

4.2.4. Nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy is a technique able to determine the atomic structures of large molecules in solution. NMR uses the fact that some atoms nuclei are naturally magnetic. This property is called spin. The atom utilized in the case of glycosphingolipids is mainly the hydrogen nucleus, 1H. The spin of the 1H nucleus can have the states, α and β, which have different energies, α having the lowest. The difference in the energy between the states increases with applied magnetic field.

By applying a radiofrequency of electromagnetic radiation, the nuclei will resonate between α and β state, yielding resonance spectra. Atoms in the surrounding area of the 1H-atoms will give local magnetic fields contrasting the applied field that can be seen in the spectra. The different frequencies gained is called chemical shifts and given in the unit; parts per million (ppm) [100].

NMR of glycosphingolipids is nowadays a non-destructive method that uses DMSO and D2O as solvents [101]. The temperature is important due to variations in chemical shifts, so references should ideally be run at the same temperature [102].

1D NMR is used to find the saccharide composition and anomeric linkages, but for determination of the carbohydrate sequences, the data from both 1D NMR and MS are combined. On the other hand, 2D NMR spectroscopy methods, as correlated spectroscopy (COSY) [103], are able to determine the connectivity between saccharaides, with the disadvantage of requiring more time.

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4.3. Flow cytometry

Flow cytometry is a method for cell counting in heterogeneous cell suspensions. The cells are recorded by light scattering and fluorescent features while moving through a laser/light beam in a liquid channel. This enables the cells to be counted, sorted, detected by size and classified by fluorescent fluorochrome-linked molecules such as antibodies. The sorting enables collection of subpopulations of cells in the samples. Multiple fluorochromes can be used at the same time, which facilitates sorting and analysis against several parameters, which are analyzed by software, displaying it in the form of single to three parameter histograms [104]. In clinical work flow cytometry is used for diagnostics and monitoring of e.g. cancer, immune deficiencies and cell-based therapies [105].

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5. Results and discussion For structural characterization of glycosphingolipids a combination of different techniques is used to get reliable information. The tools include mass spectrometry, proton NMR, thin-layer chromatography, chromatogram binding assays, as well as prior knowledge of the pathways of glycan synthesis. The identification of glycosphingolipids is a central theme in this thesis, used for description of bacterial binding receptors in Paper I and II, and used for mapping glycosphingolipid expression in Paper III and IV.

The carbohydrates expressed on cell surfaces influences the cells susceptibility to pathogens, differentiation and immune response. Thus, the characterization of glycans is important to understand these areas.

5.1. Glycosphingolipids in infection

5.1.1. Paper I: Re-definition of the carbohydrate binding specificity of Helicobacter pylori BabA adhesin.

H. pylori specialist and generalist strains BabA adhesins have different tolerance for modifications of the Leb and H type 1 epitope Fucα2Gal. Specialist strains do not tolerate any substitutions, while the generalist BabA can tolerate substitutions in 3-position with αGal as in blood group B antigens, and αGalNAc as in blood group A antigens [46]. The structural binding requirements of BabA were here further investigated.

When binding of H. pylori generalist, specialist and BabA deletion mutant strains were examined using mixtures of glycosphingolipids, an unexpected binding of specialist and generalist H. pylori to the hexaosylceramide region of porcine intestinal non-acid glycosphingolipids was found. The initial hypothesis was that the binding-active compound was a Leb hexaosylceramide, although no Leb epitopes have so far been reported in pigs. However, after isolation and characterization by mass spectrometry and NMR the binding-active glycosphingolipid was determined as Globo H hexaosylceramide (H type 4). Further binding studies demonstrated that the generalist strain, but not the specialist strain, also recognized Globo A heptaosylceramide (A type 4).

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Figure 7:. MS2 spectra of [M-H+]- ion at m/z 1014 from blood group O porcine small intestinal epithelium. C-type ions predict the structure as Fuc-Hex-HexNAc-Hex-Hex-Hex, while the 0,2A ions at m/z 792 and m/z 954 defines 1-4 linkages between saccharide 4→5 and 5→6. This suggests the Globo H pentasaccharide (Fucα2Galβ3GalNAcβ3Galα4Galβ4Glc).

The novel binding activities of BabA were most likely due to conformational similarities of the terminal disaccharides of H type 1 pentaglycosylceramide and H type 4 hexaglycosyl-ceramide, and the terminal trisaccharides of A type 1 hexaosylceramide and Globo A heptaosylceramide, respectively, as demonstrated by molecular modeling (Figure 8).

Figure 8. Minimal energy conformations predicted using CHARMm force field within the Discovery Studio molecular modeling package (Accelrys, Inc., San Diego CA), with initial points of torsion angles from literature [106, 107]. Glycosphingolipids with blood group H antigens (Left). Glycosphingolipids with blood group A antigens (Right). In addition, a number of low migrating fractions of porcine intestinal epithelium were bound by H. pylori. These compounds were identified as branched deca- and undecaglycosyl-ceramides with at least one H type 1 branch. These branched glycosphingolipids explains the H. pylori binding in the slow-migrating regions of the chromatogram binding assays. Notable is a novel undecasylceramide with one Ley on the 6-branch and a H type 1 on the 3-branch (Fucα2Galβ4(Fucα3)GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer). The results from chromatogram binding assays are summarized in Table 3.

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Table 3: Summary of the glycosphingolipid binding of H. pylori generalist and specialist strains. The BabA knock-out strain was tested to exclude non-BabA binding.

Abbreviation H. pylori J99 (generalist)

H. pylori S831 (specialist)

H. pylori J99/BabA-

A tetraosylceramide - - - Lea pentaosylceramide - - - Lex pentaosylceramide - - - H type 1 pentaosylceramide + + - H type 2 pentaosylceramide - - - H type 4 (Globo H) hexaosylceramide + + - Leb hexaosylceramide + + - Ley hexaosylceramide - - - B type 1 hexaosylceramide + - - B type 2 hexaosylceramide - - - A type 1 hexaosylceramide + - - A type 2 hexaosylceramide - - - B type 1 heptaosylceramide + - - A type 1 heptaosylceramide + - - A type 2 heptaosylceramide - - - A type 4 (Globo A) heptaosylceramide + - - A type 1 octaosylceramide + - - A type 1 nonaosylceramide + - - A type 2 nonaosylceramide - - - A type 3 nonaosylceramide - - - Lea octaosylceramide - - - Lex octaosylceramide - - - Branched H type 1 decaosylceramide + + - Branched H type 1/H type 2 decaosylceramide + + - Branched Ley/H type 1 undecaosylceramide + + - NeuGc-neolactohexaosylceramide + + +

It should be noted that Leb was the best ligand for both the generalist and specialist H. pylori strains. However, non-secretor individuals have low amounts or no Leb antigens on their epithelial surfaces, since the precursor of the Leb sequence, i.e. the H type 1 sequence, is not formed due to lack of a functional FUT2 enzyme. Still non-secretor individuals have an increased risk of peptic ulcer disease [108]. The finding that BabA also binds to GloboH offers resolution of the seeming contradiction since the Globo H sequence can still be formed by FUT1 [109, 110], and might thus function as an adhesion factor for BabA-expressing H. pylori.

Infection with Helicobacter pylori is correlated with an increased risk for gastric cancer and lymphoma, and is regarded as a paradigm for carcinogenesis mediated by an infection-induced chronic inflammation. Although the prevalence of H. pylori is decreasing in developed regions, the prevalence is still high in developing countries [39]. Furthermore, we are today facing a situation with increasing resistance against antibiotics. Novel therapeutic strategies are thus urgently needed. The BabA adhesin is an important target for future actions against H. pylori, both as vaccine candidate and as target for anti-adhesion strategies.

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5.1.2. Paper II: The repertoire of glycosphingolipids recognized by Vibrio cholerae.

V. cholerae O1 pathogenic strains of classical and El Tor biotypes were examined for binding to various glycosphingolipid mixtures, to identify potential glycan binding epitopes. Detected binding active glycosphingolipids were characterized by mass spectrometry and NMR.

Figure 9: Mass spectrometry of oligosaccharides derived from V. cholerae El tor binding glycosphingolipids of rabbit thymus. A) MS2 of [M-H+]- ion at m/z 1071 with interpretation formula. B) MS2 of [M-H+]- ion at m/z 1233 with interpretation formula. C) MS2 of [M-H+]- ion at m/z 1436 with interpretation formula.

A number of V. cholerae binding glycosphingolipids were isolated and characterized (Figure 9), and binding of the bacteria to reference glycosphingolipids was also examined (Table 4). From these experiments we were able to discern three types of glycosphingolipid binding classes.

Glycosphingolipids with a GlcNAcβ3Galβ3/4GlcNAc sequence were binding in the binding pattern called lacto/neolacto-binding (Figure 10). However certain substitutions obstruct the binding, such as terminal NeuAcα3 on neolactohexaosylceramide, or Fucα3 on any of the GlcNAcs. Some differences were seen between the lacto and neolacto sequences, the most notable were that a Fucα4 substitution is tolerated on the innermost GlcNAc for lacto core whereas not for neolacto core. The conformation and/or torsion angle may be the cause, or it could be that two different laco/neolacto binding adhesins is causing these binding patterns. This is somewhat supported by the fact that the classical strain is unable to bind lacto terminated glycosphingolipids, while binding complex neolacto glycosphingolipids. Consequently, more studies to identify the adhesin(s) involved are needed.

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Figure 10: Summary of V.cholerae El Tor binding to lacto and neolacto glycosphingolipid. The saccharide sequences in the red circles are required for high affinity binding. +++ denotes strong binding on thin-layer chromatograms while + denotes an occasional binding.

The second binding specificity is the Galα3Galα3Gal-binding. The glycosphingolipids Galα3-Galα3Galβ4GlcNAcβ4Galβ4Glcβ1Cer and Galα3Galα3Galβ4Glcβ1Cer were recognized by V. cholerae, while Galα3Galβ4GlcNAcβ4Galβ4Glcβ1Cer was not. Thus, the Galα3GalαGal sequence is the minimum binding epitope. The enzymes α1,3-galactosyltransferase or the iGb3 synthase could be producing these structures [111, 112], but these enzymes are non-functional in humans. That suggests that this binding specificity is not involved in adhesion of V. cholerae to the human small intestinal epithelium.

The third binding type is the lactosylceramide-binding. This type of binding represents binding to lactosylceramide, galactosylceramide, isoglobotriaosylceramide, gangliotriaosyl-ceramide and gangliotetraosylceramide. Binding to this battery of glycosphingolipids has previously been reported for both pathogens and indigenous flora [113]. The lactosyl-ceramide-binding does most likely not have a major role in human infection, because none of these glycosphingolipids have been found in the human intestinal epithelium [114].

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Table 4: V. cholerae El Tor binding glycosphingolipids. Bold character type designates the structural binding motifs.

Trivial name Structure I. Neolacto binding Neolactotetra Galβ4GlcNAcβ3Galβ4Glcβ1Cer GlcNAcβ3Galβ4GlcNAcβ3Galβ4Glcβ1Cer Neolactohexa Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4Glcβ1Cer B7 Galα3Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4Glcβ1Cer Neolactoocta Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4Glcβ1Cer Galα3Galβ4GlcNAcβ6(Galα3Galβ4GlcNAcβ3)Galβ4GlcNAcβ3Galβ4Glcβ1Cer II. Lacto binding Lactotetra Galβ3GlcNAcβ3Galβ4Glcβ1Cer Lacto-Lea Galβ3GlcNAcβ3Galβ3(Fucα4)GlcNAcβ3Galβ4Glcβ1Cer III. Lactosylceramide binding GalCer Galβ1Cer LacCer t18:0-h16:0-h24:0 Galβ4Glcβ1Cer Isoglobotri Galα3Galβ4Glcβ1Cer Isoglobotetra GalNAcβ3Galα3Galβ4Glcβ1Cer Gangliotri GalNAcβ4Galβ4Glcβ1Cer Gangliotetra Galβ3GalNAcβ4Galβ4Glcβ1Cer IV. Galα3Galα3Gal binding Galα3Galα3Galβ4Glcβ1Cer Galα3-B5 Galα3Galα3Galβ4GlcNAcβ3Galβ4Glcβ1Cer V. cholerae with non-functional chitin binding protein GbpA bound to glycosphingolipids in the same manner as the wild type bacteria, demonstrating that the GbpA is not involved in glycosphingolipid recognition. A recent study came to a similar conclusion when examining the binding of GbpA on glycan arrays [115].

5.2. Glycosphingolipids in differentiation

5.2.1. Paper III: Structural complexity of non-acid glycosphingolipids in human embryonic stem cells grown under feeder-free conditions.

Glycosphingolipids from two human embryonic stem cell (hESC) lines called SA121 and SA181, were characterized after culture in feeder free conditions. These culture conditions eliminated the risk of glycosphingolipid contamination from the mouse embryonic fibroblast feeder cells. The resolution of our analysis was increased by using a large amount of starting material (~1×109 cells), allowing separation of non-acid from acid glycosphingolipids, and fractioning of non-acid fractions. The large amount of starting material also enabled chromatogram binding studies.

The glycosphingolipids in the non-acid fractions were first analyzed by a thin-layer chromatogram with chemical staining which showed glycosphingolipids migrating from the monosaccharide to hexasaccharide regions. The initial chromatogram binding assays indicated the presence neolactotetraosylceramide, H type 1 pentaosylceramide, globopenta-osylceramide and Globo H pentaosylceramide.

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The non-acid fraction from each cell line were separated by Iatrobeads column chromatography into four fractions The three fractions containing fast-migrating glycosphingolipids fractions were analyzed by mass spectrometry in native form by separating on a hydrophilic interaction liquid chromatography column (polyamine II) coupled to the mass spectrometer [116, 117]. Results are summarized in Table 5.

The fractions containing slow-migrating glycosphingolipids were further tested by monoclonal antibodies in the chromatogram binding assay. A binding of antibodies against the blood group A determinant in the hexaosylceramide region was here obtained. In the slow-migrating region, glycosphingolipids were recognized by anti-Lex and a highly cross-reactive anti-Ley antibody.

These fractions were digested by Rhodococcus endoglycoceramidase and the oligosaccharides thereby released were analyzed by LC-ESI/MS using graphitized carbon columns to separate isomeric saccharides. The base peak chromatogram obtained showed molecular ions ranging from trisaccharides to hexasaccharides. Additionally, some minor oligosaccharides were detected by examination of selected ions.

MS2 of the molecular ions gave a tentative identification of globotri, lactotri, globotetra, lactotera, neolactotetra, H type 1 penta, H type 2 penta, Lex penta, globopenta, Ley hexa, globo H hexa and A type 1 hexa saccharides. The interpretation was guided by comparison of MS2 spectra and retention times of reference oligosaccharides. The identity of several of the glycosphingolipids was also confirmed by proton-NMR and chromatogram binding experiments (Table 5).

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Figure 11: MS2 of Endoglycoceramidase II yielded oligosaccharides from human embryonic stem cells. A) MS2

of the [M-H+]- ion at m/z 1217 B) MS2 of the [M-H+]- ion at m/z 1363 C) Structural interpretation formulas.

Finally, the fractions containing the slow-migrating glycosphingolipids were separated one more time with Iatrobeads chromatography, and the fractions obtained were digested by Rhodococcus endoglycoceramidase II, followed by analysis of the released oligosaccharides by LC-ESI/MS. The mass spectrometry revealed the same oligosaccharides as described above, and two additional ions at m/z 1217 and m/z 1363 were found in the two most slow-migrating fractions. By MS2 of these ions a Lex heptasaccharide (m/z 1217) and a Ley octasaccharide (m/z 1363) were tentatively identified (Figure 11). These structures could not be confirmed with NMR due to low amounts of sample but is supported by chromatogram binding assays.

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Table 5: Results from characterization by chromatogram binding assay (CBA), MS, and proton NMR, of non-acid glycosphingolipids in human embryonic stem cells.

Glycosphingolipid SA121 cell line SA181 cell line CBA MS NMR CBA MS NMR Previously found hESC glycosphingolipids Globotetraosylceramide ND + + ND + + Globopentaosylceramide/SSEA-3 + + + + + + Globo H hexaosylceramide + + + + + + Lactotetraosylceramide ND + + ND + + H type 1 pentaosylceramide + + + + + + Novel glycosphingolipids found in hESC Glucosylceramide NDa +b + ND +b + Galactosylceramide ND +b + ND +b + Lactosylceramide ND +c + ND +c + Galabiaosylceramide ND +c + ND +c + Globotriaosylceramide ND + + ND + + Lactotriaosylceramide ND + − ND + − Neolactotetraosylceramide + + − + + − H type 2 pentaosylceramide − + − − + − A type 1 hexaosylceramide + + − + + − Lex/SSEA-1 pentaosylceramide + + + + + + Ley hexaosylceramide + + − + + − Lex/SSEA-1 heptaosylceramide + + − + + − Ley octaosylceramide + + − + + −

a ND, not determined. b Glucosylceramide and galactosylceramide give identical mass spectra. c Lactosyl-ceramide and galabiaosylceramide give identical mass spectra.

Thus, several new glycosphingolipids previously not found in hESC were identified, as e.g. the blood group A type 1 hexaosylceramide. Since the ABO system is a strong immunological barrier, the blood groups should be taken into consideration when hESC are generated for future clinical applications.

5.3. Glycosphingolipids in immune recognition

5.3.1. Paper IV: Genetic basis of PX2, a recently acknowledged glycolipid blood group antigen.

This study was initiated to investigate the cause of an unexpected reaction of the plasma of a group A1B person with the P1

k phenotype with pp erythrocytes. The working hypothesis was that the p phenotype erythrocytes express a glycosphingolipid not found in P1

k/P2k

erythrocytes, and that such persons have natural antibodies against this glycosphingolipid antigen. Thorn et al. [118] have previously shown that erythrocytes of p phenotype have an increased amounts of the x2 glycosphingolipid (GalNAcβ3Galβ4GlcNAcβ3Galβ4Glcβ1Cer). This is a potential P antigen since the terminal disaccharide (GalNAcβ3Gal-R) is the same as the P glycosphingolipid. This is also supported by an earlier study that identified the x2

glycosphingolipid following observations of additional reactivity by rabbit P sera on erythrocyte membranes [119]. In order to accurately determine the role of x2 in the P1PK and GLOB system, a characterization of glycosphingolipids expressed on pp and P1

k erythrocytes was done.

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Thus, non-acid and acid glycosphingolipid fractions from human App and AP1k erythrocytes

were isolated, and after separation fractions with glycosphingolipids migrating in the diglycosylceramide region and below were obtained. These fractions were characterized by chromatogram binding studies and mass spectrometry. In a similar manner, the acid glycosphingolipid fraction from App erythrocytes was separated into the two fractions with glycosphingolipids migrating in the sialyl-neolactotetraosylceramide region and below.

The non-acid fraction from App erythrocytes was hydrolyzed by Rhodococcus endoglycoceramidase II and the oligosaccharides obtained were analyzed by LC-ESI/MS. The base peak chromatogram (Figure 12A) had a molecular ion at m/z 909, and MS2 of this ion (Fig. 12B) allowed identification of the x2 pentasaccharide (GalNAcβ3Galβ4GlcNAcβ3Gal-β4Glc).

Figure 12: A) Base peak chromatogram of glycosphingolipid-derived oligosaccharides of App erythrocytes, obtained after digestion by Rhodococcus endoglycoceramidase II. B) MS2 spectrum of the [M-H+]- ion at m/z 909, retention time 33.7 min. The spectrum displayed C-type fragment ions at m/z 382, 585 and 747 representing a pentasaccharide with (HexNAc, Hex)-HexNAc-Hex-Hex sequence. A 0,2A-type-crossring fragment was found at m/z 484, and a 0,2A3-H2O ion at m/z 466, which revealed a 4-substituted internal HexNAc. Another 0,2A-ion was found at m/z 849, with the associated 0,2A5-H2O ion at m/z 831, which demonstrated a 4-substituted Glc of the lactose unit at the reducing end. From these data the oligosaccharide was tentatively identified as the x2 pentasaccharide (GalNAcβ3Galβ4GlcNAcβ3Galβ4Glc).

Other oligosaccharide ions found in the base peak chromatogram at m/z 544, 706, 852, 1055, 1217 and 1420, were characterized as lactotri, neolactotetra, H type 2 penta, A type 2 hexa, H type 2 hepta and A type 2 octa saccharides, respectively (Figure 12A).

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The base peak chromatogram of the AP1k oligosaccharides was similar to App, with

molecular ions of lactotri, neolactotetra, H type 2 penta, A type 2 hexa, H type 2 hepta, and A type 2 octa saccharides. However, here a molecular ion at m/z 503, identifying a globo tri saccharide was also present, while no molecular ion at m/z 909 was found.

The native acid glycosphingolipid fractions from App erythrocytes were analyzed by LC-ESI/MS, with a hydrophilic interaction liquid chromatography column prior to MS. The base peak chromatogram of the first fraction was dominated by the a [M-2H+]2- ion at m/z 813, and MS2 identified a ganglioside with NeuAc-Hex-HexNAc-Hex-Hex sequence and d18:1-24:1 ceramide. Thus, sialyl-neolactotetraosylceramide was tentatively identified. A [M-2H+]2- ion at m/z 914 was also found in the base peak chromatogram. MS2 of this ion revealed a NeuAc-HexNAc-Hex-4HexNAc-Hex-Hex carbohydrate sequence and d18:1-24:1 ceramide, indicating a sialyl-x2 ganglioside. Sialyl-x2 has previously been found in human erythrocytes

[120], and α2,3-sialylation of the terminal GalNAc of x2 by ST3Gal II has been reported [121].

A [M-2H+]2- ion at m/z 959 was the major ion in the base peak chromatogram of the second acid fraction. MS2 of this ion indicated a ganglioside with NeuAc-NeuAc-Hex-HexNAc-Hex-Hex sequence and d18:1-24:1 ceramide, such as disialyl-neolactotetraosylceramide ganglioside. A minor [M-2H+]2- ion at m/z 1060 was also found in the base chromatogram. MS2 identified this as a ganglioside with NeuAc-NeuAc-HexNAc-Hex-HexNAc-Hex-Hex sequence with d18:1-24:1 ceramide, thus proposing a disialyl-x2 ganglioside.

After showing the presence of x2 pentaosylceramide among the non-acid glycosphingolipids of App erythrocytes, and the sialyl- and disialyl-x2 gangliosides among the acid glycosphingolipids, the binding of P1

k plasma and TH2 antibody (anti-x2) were tested. The P1k

plasma and the anti-x2 TH2 antibody both bound in the pentaosylceramide region of the App non-acid fraction, indicating that the P1

k plasma had anti-x2 reactivity. The P1k plasma also

bound to two gangliosides in the acid fractions from App, most likely to sialyl-x2 ganglioside and disialyl-x2 gangliosides. The TH2 antibody did not bind to the acid fractions, demonstrating that this antibody does not tolerate a substitution of sialic acid to the x2 epitope. Furthermore, no binding of TH2 antibody to the non-acid glycosphingolipids of AP1

k erythrocytes was observed.

The megakaryoblastic leukemia cell line MEG-01 was transfected with B3GALNT1 ORF and one additional cell line was mock transfected. These cell lines were used for glycosphingo-lipid extraction and partial purification, and the crude fractions obtained, containing both acid and non-acid glycosphingolipids, were tested for TH2 binding. Here, the TH2 antibody bound to the glycosphingolipids from B3GALNT1 transfected MEG-01 cells, while there was no binding to the glycosphingolipids from mock transfected MEG-01 cells (Figure 13). Similar results were seen when tested on flow cytometry with the TH2 antibody.

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A. Chemical detection B. Anti-PX2

Figure 13: Thin-layer chromatography of glycosphingolipids from B3GALNT1 and mock transfected MEG-01 cells. A) Chemical detection with anisaldehyde. B) Autoradiogram obtained by the anti-x2 antibody TH2. Lane 1, non-acid glycosphingolipids from human blood group AB erythrocytes (40 µg); lane 2, acid and non-acid glycosphingolipids of B3GALNT1 transfected cells, (10 µl/150 µl); lane 3, acid and non-acid glycosphingolipids of mock transfected cells, (10 µl/150 µl).

LC-MS analyses of the glycosphingolipid-derived oligosaccharide from Rhodococcus endoglycoceramidase II digestion of the B3GALNT1 and mock transfected MEG-01 preparations showed that both cell types had predominantly glycosphingolipids with type 2 core chains, such as Lex penta- and heptaosylceramide, neolactotetra- and hexaosylceramide. An increased intensity of the ion at m/z 909 (indicating the x2 pentasaccharide) was seen in the base peak chromatogram from the B3GALNT1 transfected cells (Figure 14, upper chart), indicating an increased production of the x2 pentaosylceramide upon B3GALNT1 transfection.

Figure 14: Base peak chromatogram from LC-ESI-MS of oligosaccharides obtained from B3GALNT1 and mock transfected MEG-01 cells. An increased intensity of the ion at m/z 909 (indicating the x2 pentasaccharide) was seen in the base peak chromatogram from the B3GALNT1 transfected cells (upper chart).

The glycosphingolipid characterization and binding experiments were supported by data from flow cytometry using the TH2 antibody. Thus, flow cytometry experiments showed that the

29

B3GALNT1 transcripts were suppressed by B3GALNT1 siRNA, while overexpression of B3GALNT1 increased x2 and P antigen levels.

Taken together the data from this study show that the x2 glycosphingolipid is lacking in individuals with mutated P synthase (β1,3GalNAc-T1), and these individuals have anti-x2 antibodies in plasma. We conclude that x2 fulfils blood group criteria and is synthesized by β1,3GalNAc-T1. It has therefore been given a new name, PX2, and promoted to join P in the GLOB blood group system.

These results will increase the general understanding of B3GALNT1 and GLOB blood group system. Furthermore it is of importance in transfusion medicine since a new blood selection principle for Pk individuals is recommended, suggesting that, if available, P1

k and P2k blood

should be used for transfusion to Pk individuals. This is however not easy due to the rarity of Pk phenotype blood [122]. As a second choice, p blood may be used despite the PX2 mismatch, since P antigen response is generally stronger.

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6. Future perspectives Data on adhesin binding specificities and glycan expression are of importance for understanding the mechanisms, pathogen susceptibilities and possible interventions for treatment. Our intention is that these studies can be used as additional knowledge for further studies in the glycobiology field.

The study of H. pylori BabA shows that the BabA binding specificity is more complex than previously known. We are currently investigating the glycosphingolipid binding specificity of a H. pylori strain expressing a BabA variant that is unable to bind Leb, but still mediates adherence of the bacterium to the human gastric epithelium. Further investigations should target more H. pylori strains to see if more variants of BabA binding can be found.

The studies of glycosphingolipid recognition of V. cholerae revealed three different glycosphingolipid binding patterns. Here, a natural continuation is to try to identify the corresponding adhesins. Currently we are examining the toxin-coregulated pilus as a potential candidate. Furthermore, the function of Galα3Galα3Gal binding is interesting since it should have no function in the infection stage, but might have a role in other interactions as e.g. biofilm formation.

The studies of glycosphingolipids in hESC revealed a complex expression that can be used for further investigations, such as examination of differentiation dependent changes in glycosphingolipid expression. The glycosphingolipid expression in hiPSC (human induced pluripotent stem cells) is currently being investigated and compared to hESC. It would also be interesting to compare glycosphingolipid expression in cancer stem cells, which is suggested to be a sub-population of some tumors. Since there are many similarities between stem cells and cancer stem cells, such as both can divide indefinitely and gives rise to heterogeneous cell populations, it is possible that there are several resemblances in glycosphingolipid expression.

The x2 antigen has been added to the GLOB blood group system and its genetic background confirmed. This gives additional tools in identifying the rare P associated blood groups, especially with the x2 specific antibody TH2. Whether the sialylated and disialylated x2 glycosphingolipids can be added to the GLOB blood group system should be further explored.

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7. Acknowledgements I would like to thank all people who has helped and encouraged me during my PhD. There are numerous of people I like to mention, hopefully I do not forget anyone.

Min handledare Susann Teneberg som har bistått med sin kunskap om glykobiologi och allt runtomkring. Tack för att du funnits där att fråga och diskutera med. Du har varit en fantastisk handledare.

Min handledare Michael Breimer, som tog mig in i glykobiologivärlden. Tack för att du lärde mig metoder inom glykosfingolipid isolering m.m.

Min handledare Anna Karlsson, tack för att du kunde hjälpa mig med min presentation.

Tack till ”min grupp”. Min kollega sen dag ett, Angela. Tack för all hjälp jag fått under åren här. Marianne, tack för din hjälp och alla intressanta diskussioner.

Nuvarande och tidigare studenter som forskat hos oss. Miralda, Benjamin, Dani, Klara, Filip och Erik tack för att ni berikat vår grupp.

Tack till mina medförfattare: Jonas Ångström, Hasse Karlsson, Omid Teymournejad, Sara Lindén, Michael Lebens, Anders Aspegren, Petter Björquist, Julia Westman, Jill Storry, Thierry Peyrard, Annika Hult, Åsa Hellberg och Martin Olsson

Tack till nuvarande och gamla medlemar i GTB gruppen. Mette och Lola tack för ni lärde mig om glykospfingolipid isolering och TLC, ni är lysande. Christel tack för assistans med administration och annat runtomkring.

Michael Lebens group. Michael, Stefan, Marcela, Susanne, Elisabeth and Anna-Karin. Thank you for all the support and knowledge I was given from your group.

Martin Olssons grupp; Martin, Julia, Jill, Annika, och Åsa, tack för sammarbetet med PX2 artikeln och hjärtliga mottagandet på mitt besök i Lund.

Tack Göran Larssons grupp för tiden på Sahlgrenska. Tack Adnan och Jonas för hjälpen med frystorkning.

Niclas Karlssons group, thank you for all the help I got in mass spectrometry. Niclas, Liquat, Jin and Samah thank you for assisting me when the mass spec is not cooperating.

To everyone at the MedKem department; The Mucin group, Sara Lindéns group, MPE core facility and Proteomics, thank you for making this such a great environment to work in.

Our “board game group”. János, Alexander, Gabor, Diarmuid and Filip, thank you for all the fun times after work.

Alla mina fantastiska vänner, tack för allt stöd och roliga stunder vi haft under denna tid.

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Min familj, även om ni kanske inte förstått exakt vad jag gör, så har ni gett mig ert fulla stöd. Ni är bäst! Tack Mamma, Pappa, Simon och Julia.

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